MicroRNAs miR-221 and miR-181c regulation of liver tumor growth and metastasis in mouse progeny from Dibenzo[def,p]chrysene dosed mothers fed a sulforaphane diet

Dibenzo[def,p]chrysene (DBC) is a polycyclic aromatic hydrocarbon (PAH) and an IARC classified 2A probable human carcinogen. Mouse progeny transplacentally exposed to DBC have been shown to develop lung and liver tumors, with liver tumors developing predominantly in males later in life. Sulforaphane (SFN) is an isothiocyanate that plays a crucial role in cellular protection from carcinogens and can be found in cruciferous vegetables. MicroRNAs (miRNAs) are a class of small, <200bp, non-coding RNAs that regulate gene expression post-transcriptionally. Emerging evidence has shown that miRNAs play critical roles in the development and progression of hepatocellular carcinoma (HCC). Pregnant mice were given one dose of DBC (15mg/kg) on gestation day (GD) 17 while fed a control or SFN diet (400ppm) starting on GD 9 and continued through weaning. Weaned offspring were maintained for 10 months and tissues analyzed for tumors. In this experiment, it was found that miR-181c was significantly up-regulated in both treatment groups. miR-221 was found to be significantly upregulated in the DBC/SFN treatment group. Proteins that are targeted by miR-181c have been shown to inactivate mechanisms which oncogenes use to metastasize in tissues. [Chen, Wang, Xu, Guo, Jiang, 2015] The upregulation of miR-181c and miR-221 suggests these miRNAs may be involved in hepatic cancer development in this model. Future research will explore candidate targets for mRNA level and protein expression thought to be regulated by these miRNAs

Antiproliferative effects of masitinib and imatinib against canine oral fibrosarcoma in vitro

Background: Canine oral fibrosarcoma (COF) is one of the most common oral tumors in dogs and carries a guarded prognosis due to a lack of effective systemic therapeutic options. Mastinib and imatinib are two commonly used tyrosine kinase inhibitors (TKIs) in veterinary oncology but their potential efficacy against COF is uncharacterized. To begin investigating the rationale for use of these TKIs against COF, the present study tested for the presence TKI targets PDGFR-α, PDGFR-β, Kit, and VEGFR-2 and examined the in vitro effects on cell viability after TKI treatment alone or with doxorubicin. Immunohistochemistry for PDGFR-α, PDGFR-β, Kit, and VEGFR-2 was performed in 6 COF tumor biopsies. Presence of these same receptors within 2 COF cell lines was probed by reverse transcription-polymerase chain reaction and, for those with mRNA detected, confirmed via western blot. Effects on cell viability were assessed using an MTS assay after masitinib or imatinib treatment alone (0-100 μM), or in combination with doxorubicin (0-3000 nM doxorubicin). Anti-PDGFRB siRNA knockdown was performed and the effect on cell viability quantified. Results: Expression of the TKI targets evaluated was similar between the 2 COF cell lines and the 6 COF tumor biopsies: PDGFR-α and PDGFR-β were detected in neoplastic cells from most COF tumor biopsies (5/6 and 6/6, respectively) and were present in both COF cell lines; KIT and KDR were not detected in any sample. Masitinib and imatinib IC50 values ranged from 7.9–33.4 μM, depending on the specific TKI and cell line tested. The addition of doxorubicin resulted in synergistic cytotoxicity with both TKIs. Anti-PDGFRB siRNA transfection reduced PDGFR-β protein expression by 77 % and 67 % and reduced cell viability by 24 % (p < 0.0001) and 28 % (0 = 0.0003) in the two cell lines, respectively. Conclusions: These results provide rationale for further investigation into the use of TKIs, possibly in combination with doxorubicin, as treatment options for COF.Keywords: Masitinib, Oral fibrosarcoma, Imatinib, Platelet-derived growth factor receptor, Do

Early transcriptome changes associated with western diet induced NASH in Ldlr−/− mice points to activation of hepatic macrophages and an acute phase response

BackgroundNonalcoholic fatty liver disease (NAFLD) is a global health problem. Identifying early gene indicators contributing to the onset and progression of NAFLD has the potential to develop novel targets for early therapeutic intervention. We report on the early and late transcriptomic signatures of western diet (WD)-induced nonalcoholic steatohepatitis (NASH) in female and male Ldlr−/− mice, with time-points at 1 week and 40 weeks on the WD. Control Ldlr−/− mice were maintained on a low-fat diet (LFD) for 1 and 40 weeks.MethodsThe approach included quantitation of anthropometric and hepatic histology markers of disease as well as the hepatic transcriptome.ResultsOnly mice fed the WD for 40 weeks revealed evidence of NASH, i.e., hepatic steatosis and fibrosis. RNASeq transcriptome analysis, however, revealed multiple cell-specific changes in gene expression after 1 week that persisted to 40 weeks on the WD. These early markers of disease include induction of acute phase response (Saa1-2, Orm2), fibrosis (Col1A1, Col1A2, TGFβ) and NASH associated macrophage (NAM, i.e., Trem2 high, Mmp12 low). We also noted the induction of transcripts associated with metabolic syndrome, including Mmp12, Trem2, Gpnmb, Lgals3 and Lpl. Finally, 1 week of WD feeding was sufficient to significantly induce TNFα, a cytokine involved in both hepatic and systemic inflammation.ConclusionThis study revealed early onset changes in the hepatic transcriptome that develop well before any anthropometric or histological evidence of NALFD or NASH and pointed to cell-specific targeting for the prevention of disease progression

Mechanism-based classification of PAH mixtures to predict carcinogenic potential

We have previously shown that relative potency factors and DNA adduct measurements are inadequate for predicting carcinogenicity of certain polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures, particularly those that function through alternate pathways or exhibit greater promotional activity compared to benzo[a]pyrene (BaP). Therefore, we developed a pathway based approach for classification of tumor outcome after dermal exposure to PAH/mixtures. FVB/N mice were exposed to dibenzo[def,p]chrysene (DBC), BaP or environmental PAH mixtures (Mix 1-3) following a two-stage initiation/promotion skin tumor protocol. Resulting tumor incidence could be categorized by carcinogenic potency as DBC>>BaP=Mix2=Mix3>Mix1=Control, based on statistical significance. Gene expression profiles measured in skin of mice collected 12 h post-initiation were compared to tumor outcome for identification of short-term bioactivity profiles. A Bayesian integration model was utilized to identify biological pathways predictive of PAH carcinogenic potential during initiation. Integration of probability matrices from four enriched pathways (p<0.05) for DNA damage, apoptosis, response to chemical stimulus and interferon gamma signaling resulted in the highest classification accuracy with leave-one-out cross validation. This pathway-driven approach was successfully utilized to distinguish early regulatory events during initiation prognostic for tumor outcome and provides proof-of-concept for using short-term initiation studies to classify carcinogenic potential of environmental PAH mixtures. These data further provide a ‘source-to outcome’ model that could be used to predict PAH interactions during tumorigenesis and provide an example of how mode-of-action based risk assessment could be employed for environmental PAH mixtures.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Oxford University Press and can be found at: http://toxsci.oxfordjournals.org/.Keywords: mixtures, toxicogenomics, skin cancer, polycyclic aromatic hydrocarbons, modelin

Nrf2 status affects tumor growth, HDAC3 gene promoter associations, and the response to sulforaphane in the colon

BACKGROUND: The dietary agent sulforaphane (SFN) has been reported to induce nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2)-dependent pathways as well as inhibiting histone deacetylase (HDAC) activity. The current investigation sought to examine the relationships between Nrf2 status and HDAC expression in preclinical and translational studies. RESULTS: Wild type (WT) and Nrf2-deficient (Nrf2(−/+)) mice were treated with the colon carcinogen 1,2-dimethylhydrazine (DMH) followed by 400 ppm SFN in the diet (n = 35 mice/group). WT mice were more susceptible than Nrf2(−/+) mice to tumor induction in the colon. Tumors from WT mice had higher HDAC levels globally and locally on genes such as cyclin-dependant kinase inhibitor 2a (Cdkn2a/p16) that were dysregulated during tumor development. The average tumor burden was reduced by SFN from 62.7 to 26.0 mm(3) in WT mice and from 14.6 to 11.7 mm(3) in Nrf2(−/+) mice. The decreased antitumor activity of SFN in Nrf2(−/+) mice coincided with attenuated Cdkn2a promoter interactions involving HDAC3. HDAC3 knockdown in human colon cancer cells recapitulated the effects of SFN on p16 induction. Human subjects given a broccoli sprout extract supplement (200 μmol SFN equivalents), or reporting more than five cruciferous vegetable servings per week, had increased p16 expression that was inversely associated with HDAC3 in circulating peripheral blood mononuclear cells (PBMCs) and in biopsies obtained during screening colonoscopy. CONCLUSIONS: Nrf2 expression varies widely in both normal human colon and human colon cancers and likely contributes to the overall rate of tumor growth in the large intestine. It remains to be determined whether this influences global HDAC protein expression levels, as well as local HDAC interactions on genes dysregulated during human colon tumor development. If corroborated in future studies, Nrf2 status might serve as a biomarker of HDAC inhibitor efficacy in clinical trials using single agent or combination modalities to slow, halt, or regress the progression to later stages of solid tumors and hematological malignancies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13148-015-0132-y) contains supplementary material, which is available to authorized users

Pathology and Viral Antigen Distribution of Lethal Pneumonia in Domestic Cats Due to Pandemic (H1N1) 2009 Influenza A Virus

A novel swine-origin H1N1 influenza A virus has been identified as the cause of the 2009 influenza pandemic in humans. Since then, infections with the pandemic (H1N1) 2009 influenza virus have been documented in a number of animal species. The first known cases of lethal respiratory disease associated with pandemic (H1N1) 2009 influenza virus infection in house pets occurred in domestic cats in Oregon. A 10-year-old, neutered male and an 8-year-old, spayed female domestic short hair cat died shortly after developing severe respiratory disease. Grossly, lung lobes of both cats were diffusely firm and incompletely collapsed. Histologically, moderate to severe, necrotizing to pyonecrotizing bronchointerstitial pneumonia was accompanied by serofibrinous exudation and hyaline membranes in the alveolar spaces. Influenza A virus was isolated from nasal secretions of the male and from lung homogenate of the female cat. Both isolates were confirmed as pandemic (H1N1) 2009 influenza virus by real-time reverse transcriptase PCR (rRT-PCR). Using immunohistochemistry, influenza A viral antigen was demonstrated in bronchiolar epithelial cells, pneumocytes and alveolar macrophages in pneumonic areas. The most likely sources of infection were people in the household with influenza-like illness or confirmed pandemic (H1N1) 2009 influenza. The two cases reported here provide, to the best of the authors’ knowledge, the first description of the pathology and viral antigen distribution of lethal respiratory disease in domestic cats after natural pandemic (H1N1) 2009 influenza virus infection, probably transmitted from humans.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The article is published by Sage Publications on behalf of the American College of Veterinary Pathologists, European College of Veterinary Pathologists, and the Japanese College of Veterinary. The published article can be found at: http://vet.sagepub.com/.Keywords: influenza, pneumonia, pandemic, pH1N1, lung, immunohistochemistry, ca

Histology of the Ovary of the Laying Hen (Gallus domesticus)

The laying hen (Gallus domesticus) is a robust animal model for epithelial ovarian cancer. The use of animal models is critical in identifying early disease markers and developing and testing chemotherapies. We describe the microscopic characteristics of the normally functioning laying hen ovary and proximal oviduct to establish baselines from which lesions associated with ovarian cancer can be more readily identified. Ovaries and oviducts were collected from 18-month-old laying hens (n = 18) and fixed in 10% neutral buffered formalin. Hematoxylin- and eosin-stained sections were examined by light microscopy. Both post-ovulatory follicular regression and atresia of small follicles produce remnant clusters of vacuolated cells with no histological evidence that scar tissue persists. Infiltrates of heterophils are associated with atresia of small follicles, a relationship not previously documented in laying hen ovaries. Because these tissues can be mistaken for cancerous lesions, we present a detailed histological description of remnant Wolffian tissues in the laying hen ovary. Immunohistochemical staining for pancytokeratin produced a positive response in ovarian surface epithelium and staining for vimentin produced a positive response in granulosa cells of follicles. Epithelial cells lining glands of the remnant epoöphoron had a positive response to both pancytokeratin and vimentin, a result also observed in women

LöhrChristianeVeterinaryMedicineCharacterizationMicroanatomyHistopathology.pdf

From 2002 to 2007, 101 camelid abortions and stillbirths were submitted to the Veterinary Diagnostic Laboratory at Oregon State University (84 alpacas, 13 llamas, four unknown). For most of the cases (n=67) a cause was not determined by routine testing. Eighty-five submissions included placenta for microscopic examination of which 55 were from abortions to unknown causes (idiopathic). Microscopic features of placentas from abortion/stillbirth were compared with those from 19 camelids delivered normally (six alpacas, 12 llamas, one unknown) and those from four alpaca fetuses of known gestational age collected during the dam’s necropsy. The most common microscopic findings in abortion/stillbirth placentas were mineralization (n=57) and mucinous edema (n=27) of the chorioallantoic stroma. One or more of these features were also observed in 22/23 placentas from normal pregnancies/deliveries and therefore interpreted as incidental findings. The comparison of alpaca placentas after matching for gestational parameters (crown rump length, weight, days of gestation; n=41) revealed hypoplasia of placental villi in 5/22 idiopathic abortions and in one abortion due to umbilical torsion, and was suspected in an additional six abortions of unknown and two abortions of known cause. The identified villous hypoplasia is assumed to have resulted in placental insufficiency. When placental insufficiency is included as cause, idiopathic abortions are reduced from 66.2% to 47.9% of alpaca cases with histopathological examination of placenta and from 66.3 to 52.5% of alpaca and llama abortions overall. This study also permitted the generation of a linear regression curve correlating alpaca fetal crown-rump length with fetal age

LöhrChristianeVeterinaryMedicineCharacterizationMicroanatomyHistopathology_Figure1.tif

From 2002 to 2007, 101 camelid abortions and stillbirths were submitted to the Veterinary Diagnostic Laboratory at Oregon State University (84 alpacas, 13 llamas, four unknown). For most of the cases (n=67) a cause was not determined by routine testing. Eighty-five submissions included placenta for microscopic examination of which 55 were from abortions to unknown causes (idiopathic). Microscopic features of placentas from abortion/stillbirth were compared with those from 19 camelids delivered normally (six alpacas, 12 llamas, one unknown) and those from four alpaca fetuses of known gestational age collected during the dam’s necropsy. The most common microscopic findings in abortion/stillbirth placentas were mineralization (n=57) and mucinous edema (n=27) of the chorioallantoic stroma. One or more of these features were also observed in 22/23 placentas from normal pregnancies/deliveries and therefore interpreted as incidental findings. The comparison of alpaca placentas after matching for gestational parameters (crown rump length, weight, days of gestation; n=41) revealed hypoplasia of placental villi in 5/22 idiopathic abortions and in one abortion due to umbilical torsion, and was suspected in an additional six abortions of unknown and two abortions of known cause. The identified villous hypoplasia is assumed to have resulted in placental insufficiency. When placental insufficiency is included as cause, idiopathic abortions are reduced from 66.2% to 47.9% of alpaca cases with histopathological examination of placenta and from 66.3 to 52.5% of alpaca and llama abortions overall. This study also permitted the generation of a linear regression curve correlating alpaca fetal crown-rump length with fetal age